The study of what appeared to be quite diverse physiological reactions led to the discovery of nitric oxide (N=O) as a mammalian metabolite that plays a critical role in cellular communication as well as other important functions. The investigation of the apparent synthesis of nitrate (NO3-) by mammalian cells revealed that, in macrophages, N=O was an intermediate in the production of both NO3- and nitrite (NO2-) from L-arginine. Nitric oxide appears to play a critical role in cellular communication as well as other important functions. Therefore exposure to N=O occurs through normal intermediary metabolism. Consequently the exposure to N2O3, agents capable of reacting with secondary amines to form N-nitrosamines, occurs via endogenous processes as well. The significance of this metabolic formation of N-nitrosating agents to the endogenous synthesis of N-nitrosamines is an underlying theme of the entire program project. The cell-specific origin of endogenously formed N-nitrosating agents will be very important in determining the potential for this pathway to form N-nitrosamines and if specific cellular and/or pathologic processes influence the formation of N- nitrosamines. The specific aims in this continuation of our studies will expand further cell-types that express this pathway and the conditions that lead to its expression. These questions will be addressed at the level of the products of the reaction, the enzyme responsible for N=O synthesis, and genes and message for the relevant proteins. In addition there is a critical need for a human cell culture model for this pathway and as yet none exists. It is an important gap and one that this proposal intends to fill. Certain pathophysiological states, such as colitis and wound healing involve the macrophage. Additionally, L-arginine metabolism is known to participate in wound healing and repair and so we will use model to probe the involvement of this pathway in this important process. Virtually nothing is known about the chemistry of the immediate precursor to N=O and this proposal will address this as well.